Aim:To examine the transfection of exogenous genes into chick embryos,applying the characteristics of avianleukosis vires(ALV)-induced chicken B cell line DT40 to the production of chimeric birds.Methods:The DT40cells...Aim:To examine the transfection of exogenous genes into chick embryos,applying the characteristics of avianleukosis vires(ALV)-induced chicken B cell line DT40 to the production of chimeric birds.Methods:The DT40cells incorporated with exogenous gene(lacZ constructs encoding Escherichia coliβ-galactosidase:β-gal)were intro-duced into chick embryos by the injection of cells into stage X blastoderm.Manipulated eggs were incubated for 3(trial1)or 6(trial 2)days,and the expression of lacZ DNA was detected by a histochemical staining method ofβ-galactosi-dase and polymerase chain reaction(PCR)analysis.Results:The survival rates of the manipulated embryos incu-bated for 3 days(stage 18-20:trial 1)and 6 days(stage 28,30:trial 2)were about 42%and 38%,respectively.The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60%and 23%,respectively,forthe survived embryos.Conclusion:The rate of embryonic viability and expression rate of introduced genes were notso high,but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chickembryos.展开更多
Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen ...Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 ℃ for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light. Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L -5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.展开更多
An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, t...An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chickswere raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female trans-genic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination ac-cording to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the em-bryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As aresult, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts fromembryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of in-jected MiwZ in the extracts from sperm was also confirmed. The exogenous gene introduced into the GCR migrated suc-cessfully to the gonad resulting in its incorporation into the offspring and spermatozoa of transgenic chickens. (Asian JAndrol 1999 Sep ; 1: 139 - 144)展开更多
基金financially supported by the Ministry of Education,Science and Culture,Japanthe Society for the Promotion of Science(JSPS)+1 种基金the Sumitomo Foundationthe Nissan Science Foundation
文摘Aim:To examine the transfection of exogenous genes into chick embryos,applying the characteristics of avianleukosis vires(ALV)-induced chicken B cell line DT40 to the production of chimeric birds.Methods:The DT40cells incorporated with exogenous gene(lacZ constructs encoding Escherichia coliβ-galactosidase:β-gal)were intro-duced into chick embryos by the injection of cells into stage X blastoderm.Manipulated eggs were incubated for 3(trial1)or 6(trial 2)days,and the expression of lacZ DNA was detected by a histochemical staining method ofβ-galactosi-dase and polymerase chain reaction(PCR)analysis.Results:The survival rates of the manipulated embryos incu-bated for 3 days(stage 18-20:trial 1)and 6 days(stage 28,30:trial 2)were about 42%and 38%,respectively.The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60%and 23%,respectively,forthe survived embryos.Conclusion:The rate of embryonic viability and expression rate of introduced genes were notso high,but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chickembryos.
文摘Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 ℃ for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light. Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L -5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.
文摘An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chickswere raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female trans-genic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination ac-cording to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the em-bryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As aresult, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts fromembryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of in-jected MiwZ in the extracts from sperm was also confirmed. The exogenous gene introduced into the GCR migrated suc-cessfully to the gonad resulting in its incorporation into the offspring and spermatozoa of transgenic chickens. (Asian JAndrol 1999 Sep ; 1: 139 - 144)
